The monokaryotic filamentous fungus Ustilago sp. HFJ311 promotes plant growth and reduces Cd accumulation by enhancing Fe transportation and auxin biosynthesis.

Infection with smut fungus like Ustilago maydis decreases crop yield via inducing gall formation. However, the in vitro impact of Ustilago spp. on plant growth and stress tolerance remains elusive. This study investigated the plant growth promotion and cadmium stress mitigation mechanisms of a filamentous fungus discovered on a cultural medium containing 25 µM CdCl2. ITS sequence alignment revealed 98.7 % similarity with Ustilago bromivora, naming the strain Ustilago sp. HFJ311 (HFJ311). Co-cultivation with HFJ311 significantly enhanced the growth of various plants, including Arabidopsis, tobacco, cabbage, carrot, rice, and maize, and improved Arabidopsis tolerance to abiotic stresses like salt and metal ions. HFJ311 increased chlorophyll and Fe contents in Arabidopsis shoots and enhanced root-to-shoot Fe translocation while decreasing root Fe concentration by approximately 70 %. Concurrently, HFJ311 reduced Cd accumulation in Arabidopsis by about 60 %, indicating its potential for bioremediation in Cd-contaminated soils. Additionally, HFJ311 stimulated IAA concentration by upregulating auxin biosynthesis genes. Overexpression of the Fe transporter IRT1 negated HFJ311's growth-promotion effects under Cd stress. These results suggest that HFJ311 stimulates plant growth and inhibits Cd uptake by enhancing Fe translocation and auxin biosynthesis while disrupting Fe absorption. Our findings offer a promising bioremediation strategy for sustainable agriculture and food security.

Root radial apoplastic transport contributes to shoot cadmium accumulation in a high cadmium-accumulating rice line.

Radial transport of cadmium (Cd) in roots governs the amount of Cd loaded into xylem vessels, where Cd ions are translocated upward into shoots, while the mechanism of differential Cd radial transport between the high Cd-accumulating rice line Lu527-8 and the normal rice line Lu527-4 remains ambiguous. A higher Cd distribution in cross sections and root apoplast and higher bypass flow of Cd were found in Lu527-8, explaining a greater Cd translocation through the apoplastic pathway. The lower relative area of the epidermis and the constant relative area of the cortex in Lu527-8 opened-up root radial transport for Cd. Deposition of apoplastic barriers (Casparian strips and suberin lamellae) was stimulated by Cd, which effectively prevented Cd from entering the stele through the apoplastic pathway. In Lu527-8, apoplastic barriers were further from the root apex with lower expression of genes responsible for biosynthesis of Casparian strips and suberin lamellae, enhancing radial transport of Cd. Our data revealed that the higher radial apoplastic transport of Cd played an integral role in Cd translocation, contributed to a better understanding of the mechanism involved in high Cd accumulation in Lu527-8 and helped achieve the practical application of phytoextraction.

Hydrogen peroxide mediates cadmium accumulation in the root of a high cadmium-accumulating rice (Oryza sativa L.) line.

Hydrogen peroxide (H2O2) is a vital signaling molecule in response to cadmium (Cd) stress in plants. However, the role of H2O2 on Cd accumulation in root of different Cd-accumulating rice lines remains unclear. Exogenous H2O2 and 4-hydroxy-TEMPO (H2O2 scavenger) were applied to investigate the physiological and molecular mechanisms of H2O2 on Cd accumulation in the root of a high Cd-accumulating rice line Lu527-8 through hydroponic experiments. Interestingly, it was found Cd concentration in the root of Lu527-8 increased significantly when exposed to exogenous H2O2, while reduced significantly when exposed to 4-hydroxy-TEMPO under Cd stress, proving the role of H2O2 in regulating Cd accumulation in Lu527-8. Lu527-8 showed more Cd and H2O2 accumulation in the roots, along with more Cd accumulation in cell wall and soluble fraction, than the normal rice line Lu527-4. In particular, more pectin accumulation, especially low demethylated pectin, was observed in the root of Lu527-8 when exposed to exogenous H2O2 under Cd stress, resulting in more negative functional groups with greater capacity to binding Cd in the root cell wall of Lu527-8. It indicated that H2O2-induced cell wall modification and vacuolar compartmentalization contributes greatly to more Cd accumulation in the root of the high Cd-accumulating rice line.

UCP1 and AOX1a contribute to regulation of carbon and nitrogen metabolism and yield in Arabidopsis under low nitrogen stress.

Nitrogen (N) availability is a critical factor for plant development and crop yield, and it closely correlates to carbon (C) metabolism. Uncoupling protein (UCP) and alternative oxidase (AOX) exhibit a strong correlation with N and C metabolism. Here, we investigated the functions of UCP1 and AOX1a using their mutants and complementation lines in Arabidopsis adaptation to low N. Low N markedly increased AOX1a and UCP1 expression, alternative pathway capacity and UCP activity. Eight-day-old aox1a/ucp1 seedlings were more sensitive to low N than Col-0 and single mutants, exhibiting lower primary root length and higher anthocyanin accumulation. The net photosynthetic rate, electron transport rate, PSII actual photochemical efficiency, stomatal conductance and carboxylation efficiency were markedly decreased in ucp1 and aox1a/ucp1 compared to those in Col-0 and aox1a under low N stress; comparatively, chlorophyll content and non-photochemical quenching coefficient were the lowest and highest in aox1a/ucp1, respectively. Nitrate acquisition rate was accelerated in aox1a/ucp1, but its transport activity was decreased, which resulted in low nitrate content and nitrate reductase activity under low N condition. The C/N ratio in seeds, but not in leaves, is higher in aox1a/ucp1 than that in Col-0, aox1a and ucp1 under low N condition. RNA-seq analysis revealed that many genes involved in photosynthesis and C/N metabolism were markedly down-regulated in aox1a/ucp1 under low N stress. These results highlight the key roles of UCP1 and AOX1a in modulating photosynthetic capacity, C/N assimilation and distribution under low N stress.

PIF4-PAP1 interaction affects MYB-bHLH-WD40 complex formation and anthocyanin accumulation in Arabidopsis.

Anthocyanin accumulation is a marked phenotype of plants under environmental stresses. PHYTOCHROME-INTERACTING FACTORs (PIFs) are involved in environment-induced anthocyanin biosynthesis through interacting with the MYB-bHLH-WD40 (MBW) complex. However, the molecular mechanism of this interaction remains unclear. The present study demonstrated that PIF3 and PIF5 can slightly repress anthocyanin accumulation under NaCl, low nitrogen (-N), or 6-BA treatments; in contrast, PIF4 can significantly repress anthocyanin accumulation. Bimolecular fluorescence complementation and yeast two-hybrid assays showed that PIF4 directly interacts with PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1), a MYB transcription factor in the MBW complex. Further analysis revealed that the active phytochrome binding (APB) domain in the N terminus of PIF4 is necessary for the interaction between PIF4 and PAP1. Yeast three-hybrid analysis showed that PIF4 competes with TRANSPARENT TESTA 8 (TT8) to bind PAP1, thereby interfering with the regulation of the MBW protein complex in anthocyanin synthesis. Consistently, the anthocyanin content in pap1-D/35S::PIF4 and 35S::PAP1/35S::PIF4 seedlings was markedly lower than that in pap1-D and 35S::PAP1 under 6-BA, MeJA, -N, and NaCl stresses, implying that overexpression of PIF4 suppresses anthocyanin accumulation in pap1-D and 35S::PAP1. Thus, PIF4 is genetically epistatic to PAP1. Taken together, PIF4 plays a negative role in modulating anthocyanin biosynthesis in Arabidopsis under different stress environments, and PIF4 interacts with PAP1 to affect the integrity of the MBW complex.

Integrated Physiological, Transcriptomic, and Metabolomic Analyses Revealed Molecular Mechanism for Salt Resistance in Soybean Roots.

Salinity stress is a threat to yield in many crops, including soybean (Glycine max L.). In this study, three soybean cultivars (JD19, LH3, and LD2) with different salt resistance were used to analyze salt tolerance mechanisms using physiology, transcriptomic, metabolomic, and bioinformatic methods. Physiological studies showed that salt-tolerant cultivars JD19 and LH3 had less root growth inhibition, higher antioxidant enzyme activities, lower ROS accumulation, and lower Na+ and Cl- contents than salt-susceptible cultivar LD2 under 100 mM NaCl treatment. Comparative transcriptome analysis showed that compared with LD2, salt stress increased the expression of antioxidant metabolism, stress response metabolism, glycine, serine and threonine metabolism, auxin response protein, transcription, and translation-related genes in JD19 and LH3. The comparison of metabolite profiles indicated that amino acid metabolism and the TCA cycle were important metabolic pathways of soybean in response to salt stress. In the further validation analysis of the above two pathways, it was found that compared with LD2, JD19, and LH3 had higher nitrogen absorption and assimilation rate, more amino acid accumulation, and faster TCA cycle activity under salt stress, which helped them better adapt to salt stress. Taken together, this study provides valuable information for better understanding the molecular mechanism underlying salt tolerance of soybean and also proposes new ideas and methods for cultivating stress-tolerant soybean.

Alternative Pathway Is Involved in Hydrogen Peroxide-Enhanced Cadmium Tolerance in Hulless Barley Roots.

Hulless barley, grown in the Qinghai Tibet Plateau, has a wide range of environmental stress tolerance. Alternative pathway (AP) and hydrogen peroxide (H2O2) are involved in enhancing plant tolerance to environmental stresses. However, the relationship between H2O2 and AP in hulless barley tolerance to cadmium (Cd) stress remains unclear. In the study, the role and relationship of AP and H2O2 under Cd stress were investigated in hulless barley (Kunlun14) and common barley (Ganpi6). Results showed that the expression level of alternative oxidase (AOX) genes (mainly AOX1a), AP capacity (Valt), and AOX protein were clearly induced more in Kunlun14 than in Ganpi 6 under Cd stress; moreover, these parameters were further enhanced by applying H2O2. Malondialdehyde (MDA) content, electrolyte leakage (EL) and NAD(P)H to NAD(P) ratio also increased in Cd-treated roots, especially in Kunlun 14, which can be markedly alleviated by exogenous H2O2. However, this mitigating effect was aggravated by salicylhydroxamic acid (SHAM, an AOX inhibitor), suggesting AP contributes to the H2O2-enhanced Cd tolerance. Further study demonstrated that the effect of SHAM on the antioxidant enzymes and antioxidants was minimal. Taken together, hulless barley has higher tolerance to Cd than common barley; and in the process, AP exerts an indispensable function in the H2O2-enhanced Cd tolerance. AP is mainly responsible for the decrease of ROS levels by dissipating excess reducing equivalents.

The response mechanism to salt stress in Arabidopsis transgenic lines over-expressing of GmG6PD.

Glucose-6-phosphate dehydrogenase (G6PD or G6PDH) plays an important role in response to salt stress in plants. However, much less is known about G6PD proteins in soybean (Glycine max L.). Here, we found that a soybean cytosolic G6PD gene, GmG6PD7, was induced by NaCl. We generated Arabidopsis transgenic lines overexpressing GmG6PD7. The seed germination rate and primary root length of Arabidopsis thaliana over-expressing GmG6PD7 under NaCl treatment were enhanced. Salt stress induced an obvious increase of the total and cytosolic G6PD activity and the marked decrease of ROS levels in the transgenic plants. At the same time, over-expressing GmG6PD7 in Arabidopsis affected the glutathione and NADPH level and activated ROS scavengers, suggesting that GmG6PD7 contributes to increase salinity tolerance by decreasing ROS accumulation. What's more, we found GmG6PD7 overexpression led to the up-regulation of abscisic acid (ABA) degradation gene and the down-regulation of ABA synthesis and ABA-responsive genes, which finally reduced ABA content to improve seed germination rate under salinity stress. It was noteworthy that GmG6PD7 can rescue the seed and root phenotype of Arabidopsis cytosolic G6PD mutant (Atg6pd5 and Atg6pd6) under salt stress, suggesting cytosolic G6PD may have a conserved function in soybean and Arabidopsis.

Identification, Characterization, and Stress Responsiveness of Glucose-6-phosphate Dehydrogenase Genes in Highland Barley.

G6PDH provides intermediate metabolites and reducing power (nicotinamide adenine dinucleotide phosphate, NADPH) for plant metabolism, and plays a pivotal role in the cellular redox homeostasis. In this study, we cloned five G6PDH genes (HvG6PDH1 to HvG6PDH5) from highland barley and characterized their encoded proteins. Functional analysis of HvG6PDHs in E. coli showed that HvG6PDH1 to HvG6PDH5 encode the functional G6PDH proteins. Subcellular localization and phylogenetic analysis indicated that HvG6PDH2 and HvG6PDH5 are localized in the cytoplasm, while HvG6PDH1, HvG6PDH3, and HvG6PDH4 are plastidic isoforms. Analysis of enzymatic activities and gene expression showed that HvG6PDH1 to HvG6PDH4 are involved in responses to salt and drought stresses. The cytosolic HvG6PDH2 is the major isoform against oxidative stress. HvG6PDH5 may be a house-keeping gene. In addition, HvG6PDH1 to HvG6PDH4 and their encoded enzymes responded to jasmonic acid (JA) and abscisic acid (ABA) treatments, implying that JA and ABA are probably critical regulators of HvG6PDHs (except for HvG6PDH5). Reactive oxygen species analysis showed that inhibition of cytosolic and plastidic G6PDH activities leads to increased H2O2 and O2- contents in highland barley under salt and drought stresses. These results suggest that G6PDH can maintain cellular redox homeostasis and that cytosolic HvG6PDH2 is an irreplaceable isoform against oxidative stress in highland barley.

Alternative Pathway is Involved in Nitric Oxide-Enhanced Tolerance to Cadmium Stress in Barley Roots.

Alternative pathway (AP) has been widely accepted to be involved in enhancing tolerance to various environmental stresses. In this study, the role of AP in response to cadmium (Cd) stress in two barley varieties, highland barley (Kunlun14) and barley (Ganpi6), was investigated. Results showed that the malondialdehyde (MDA) content and electrolyte leakage (EL) level under Cd stress increased in two barley varieties. The expressions of alternative oxidase (AOX) genes (mainly AOX1a), AP capacity (Valt), and AOX protein amount were clearly induced more in Kunlun14 under Cd stress, and these parameters were further enhanced by applying sodium nitroprussid (SNP, a NO donor). Moreover, H2O2 and O2- contents were raised in the Cd-treated roots of two barley varieties, but they were markedly relieved by exogenous SNP. However, this mitigating effect was aggravated by salicylhydroxamic acid (SHAM, an AOX inhibitor), suggesting that AP contributes to NO-enhanced Cd stress tolerance. Further study demonstrated that the effect of SHAM application on reactive oxygen species (ROS)-related scavenging enzymes and antioxidants was minimal. These observations showed that AP exerts an indispensable function in NO-enhanced Cd stress tolerance in two barley varieties. AP was mainly responsible for regulating the ROS accumulation to maintain the homeostasis of redox state.

Cytosolic Glucose-6-Phosphate Dehydrogenase Is Involved in Seed Germination and Root Growth Under Salinity in Arabidopsis.

Glucose-6-phosphate dehydrogenase (G6PDH or G6PD) is the key regulatory enzyme in the oxidative pentose phosphate pathway (OPPP). The cytosolic isoforms including G6PD5 and G6PD6 account for the major part of the G6PD total activity in plant cells. Here, we characterized the Arabidopsis single null mutant g6pd5 and g6pd6 and double mutant g6pd5/6. Compared to wild type, the mutant seeds showed a reduced germination rate and root elongation under salt stress. The seeds and seedlings lacking G6PD5 and G6PD6 accumulate more reactive oxygen species (ROS) than the wild type under salt stress. Cytosolic G6PD (cy-G6PD) affected the expression of NADPH oxidases and the G6PD enzymatic activities in the mutant atrbohD/F, in which the NADPH oxidases genes are disrupted by T-DNA insertion and generation of ROS is inhibited, were lower than that in the wild type. The NADPH level in mutants was decreased under salt stress. In addition, we found that G6PD5 and G6PD6 affected the activities and transcript levels of various antioxidant enzymes in response to salt stress, especially the ascorbate peroxidase and glutathione reductase. Exogenous application of ascorbate acid and glutathione rescued the seed and root phenotype of g6pd5/6 under salt stress. Interestingly, the cytosolic G6PD negatively modulated the NaCl-blocked primary root growth under salt stress in the root meristem and elongation zone.

Involvement of G6PD5 in ABA response during seed germination and root growth in Arabidopsis.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PDH or G6PD) functions in supply of NADPH, which is required for plant defense responses to stresses. However, whether G6PD functions in the abscisic acid (ABA) signaling pathway remains to be elucidated. In this study, we investigated the involvement of the cytosolic G6PD5 in the ABA signaling pathway in Arabidopsis. RESULTS: We characterized the Arabidopsis single null mutant g6pd5. Phenotypic analysis showed that the mutant is more sensitive to ABA during seed germination and root growth, whereas G6PD5-overexpressing plants are less sensitive to ABA compared to wild type (WT). Furthermore, ABA induces excessive accumulation of reactive oxygen species (ROS) in mutant seeds and seedlings. G6PD5 participates in the reduction of H2O2 to H2O in the ascorbate-glutathione cycle. In addition, we found that G6PD5 suppressed the expression of Abscisic Acid Insensitive 5 (ABI5), the major ABA signaling component in dormancy control. When G6PD5 was overexpressed, the ABA signaling pathway was inactivated. Consistently, G6PD5 negatively modulates ABA-blocked primary root growth in the meristem and elongation zones. Of note, the suppression of root elongation by ABA is triggered by the cell cycle B-type cyclin CYCB1. CONCLUSIONS: This study showed that G6PD5 is involved in the ABA-mediated seed germination and root growth by suppressing ABI5.

Alternative respiration pathway is involved in the response of highland barley to salt stress.

KEY MESSAGE: Alternative respiration pathway is involved in the response of highland barley to salt stress. The response of two barley seedlings to salt stress was investigated. Results showed that the growth of highland barley (Kunlun 14) and barley (Ganpi 6) had no obvious difference under low concentrations (50, 100 and 200 mM) of NaCl treatment. However, high concentrations of NaCl treatment (300 and 400 mM) severely affected the growth of two barley cultivars. Under 300 mM NaCl treatment, the fresh weight, relative water content (RWC), pigments and K+ content reduced more in Ganpi 6 than in Kunlun 14. In contrast, the electrolyte leakage and the content of MDA, Na+, H2O2 and O2- increased more in Ganpi 6 than in Kunlun 14. The gene expression of AOX1a, HvNHX1, HvNHX3, HvHVP1, HvHVA, H+-ATPase, the alternative respiration capacity (Valt) and the enzymatic activity of SOD, POD, CAT, APX and H+-ATPase increased more in Kunlun14 than in Ganpi6 under 300 mM NaCl treatment, whereas the cytochrome respiration capacity (Vcyt) decreased similarly in both barley cultivars. Western blot analysis showed that the protein level of the alternative oxidase (AOX) increased more in Kunlun 14 than in Ganpi 6 under 300 mM NaCl treatment. Inhibition of the alternative respiration by salicylhydroxamic acid (SHAM) decreased the fresh weight, K+ content, Valt, H+-ATPase activity and the gene expression of AOX1a, HvNHX1, HvNHX3, HvHVP1, HvHVA, H+-ATPase, but increased the electrolyte leakage, MDA and Na+ content in both cultivars under 300 mM NaCl treatment. In short, alternative respiration is involved in the tolerance of highland barley to salt stress.